The majority of vertebrate cell cultures in vitro are grown as two-dimensional monolayers on an artificial substrate bathed in nutrient medium. The nature of the substrate on which the monolayers grow may be solid, such as plastic, or semisolid gels, such as collagen or agar. Disposable plastics have become the preferred substrate used in modern-day tissue or cell culture.
While the growth of cells in two dimensions is a convenient method for preparing, observing and studying cells in culture, allowing a high rate of cell proliferation, it lacks the cell-cell and cell-matrix interactions characteristic of whole tissue in vivo. In order to study such functional and morphological interactions, a few investigators have explored the use of three-dimensional substrates such as collagen gel (Douglas et al., 1980, In Vitro 16:306-312; Yang et al., 1979, Proc. Natl. Acad. Sci. 76:3401; Yang et al., 1980, Proc. Natl. Acad. Sci. 77:2088-2092; Yang et al., 1981, Cancer Res. 41:1021-1027); cellulose sponge, alone (Leighton et al., 1951, J. Natl. Cancer Inst. 12:545-561) or collagen coated (Leighton et al., 1968, Cancer Res. 28:286-296); or a gelatin sponge, Gelfoam (Sorour et al., 1975, J. Neurosurg. 43:742-749).
In general, these three-dimensional substrates are inoculated with the cells to be cultured. Many of the cell types have been reported to penetrate the matrix and establish a "tissue-like" histology. For example, three-dimensional collagen gels have been utilized to culture breast epithelium (Yang et al., 1981, Cancer Res. 41:1021-1027) and sympathetic neurons (Ebendal, 1976, Exp. Cell Res. 98:159-169). Additionally, various attempts have been made to regenerate tissue-like architecture from dispersed monolayer cultures. Kruse and Miedema (1965, J. Cell Biol. 27:273) reported that perfused monolayers could grow to more than ten cells deep and organoid structures can develop in multilayered cultures if kept supplied with appropriate medium (see also Schneider et al., 1963, Exp. Cell Res. 30:449-459 and Bell et al., 1979, Proc. Natl. Acad. Sci. USA 76:1274-1279); Green (1978, Science 200:1385-1388) has reported that human epidermal kerotinocytes may form dematoglyphs (friction ridges) if kept for several weeks without transfer; Folkman and Haudenschild (1980, Nature 288:551-556) reported the formation of capillary tubules in cultures of vascular endothelial cells cultured in the presence of endothelial growth factor and medium conditioned by tumor cells; and Sirica et al. (1979, Proc. Natl. Acad. Sci. U.S.A. 76:283-287; 1980, Cancer Res. 40:3259-3267) maintained hepatocytes in primary culture for about 10-13 days on nylon meshes coated with a thin layer of collagen. However, the long term culture and proliferation of cells in such systems has not been achieved.
Indeed, the establishment of long term culture of tissues such as bone marrow had been attempted, but, overall, the results were disappointing, in that although a stromal cell layer containing different cell types was rapidly formed, significant hematopoiesis could not be maintained for any protracted period of time. (For review see Dexter et al., In Long Term Bone Marrow Culture, 1984, Alan R. Liss, Inc., pp.57-96).
U.S. Pat. No. 4,721,096, issued Jan. 26, 1988, and 5,032,508, issued Jul. 16, 1991, both by Naughton et al., disclose a three-dimensional cell culture system which, for the first time, achieves long-term proliferation of cells, including skin and bone marrow, as well as other tissues. Using this system, a living stromal tissue is prepared in vitro by allowing stromal cells and the connective tissue proteins they naturally secrete to attach to and substantially envelop a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces. The stromal cells bridge the interstitial spaces of the framework, thereby creating a living cellular matrix on which may be seeded parenchymal cells such as hematopoietic cells, hepatocytes, melanocytes, keratinocytes, etc. The resulting cultures give rise to "tissue equivalents" which functionally and histologically resemble naturally occurring tissues. The present invention constitutes an improvement of the three-dimensional cell and tissue culture system described in U.S. Pat. No. 4,721,096 and 5,032,508, both of which are incorporated by reference herein in their entirety.